Novel N-2-(Furyl)-2-(chlorobenzyloxyimino) Ethyl Piperazinyl Quinolones: Synthesis, Cytotoxic Evaluation and Structure-Activity Relationship.

Quinolone antibacterials are one of the most important classes of pharmacological agents known as potent inhibitors of bacterial DNA gyrase and topoisomerase IV that efficiently inhibit DNA replication and transcription by generating several double-stranded DNA break. Some quinolone derivatives demonstrated inhibitory potential against eukaryote topoismarase II and substantial dose-dependent cytotoxic potential against some cancerous cells. In present study, synthesis and cytotoxic activity evaluation of new series of N-pipearzinyl quinolones containing N-2-(furyl-2 or 3-yl)-2-(chlorobenzyloxyimino) ethyl moiety 7a-i have been studied. Reaction of quinolone, with 2-bromo-1-(furan-2 or 3-yl)ethanone-O-substituted chlorobenzyloxime in DMF in presence of NaHCO3 at room temperature, gave the title compounds N-2-(furan-2 or 3-yl)-2-(chlorobenzyloxyiminoethyl) quinolone 7a-i. Synthesized compounds were further evaluated in-vitro against three human breast tumor cell lines. Preliminary screening indicated that compound 7 g demonstrated significant growth inhibitory potential against all evaluated cell lines. The results of structure-activity relationship study exhibited that quinolone derivatives are superior in cytotoxic potential compared to 1, 8-naphthyridone series. Furthermore, ethyl quinolone derivatives were more potent cytotoxic agents comparing with cyclopropyl quinolones.


Experimental
Chemistry A Kofler hot stage apparatus was used for the measurement of reported melting. The IR spectra were recorded on a Nicollet FT-IR Magna 550 spectrometer. The 1 HNMR spectra were recorded on Bruker FT-500 MHz spectrometer and chemical shifts (δ) are reported in ppm relative to internal tetramethylsilane. Mass spectra were recorded on an Agilent Technology (HP) mass spectrometer operating at an ionisation potential of 70 eV. Analytical thin layer chromatography (TLC) on Merck silicagel 60 F254 plates using various mobile phases of different polarities was performed in order to confirm the purity of final products.
inhibit DNA replication and transcription by generating several double-stranded DNA break (4-6). Several researches have indicated that some quinolone derivatives display inhibitory potential against eukaryote topoismarase II. Therefore, in addition to antibacterial activity, some member of quinolones demonstrated substantial dose-dependent growth inhibitory potential against some cancerous cells (7-10). Moreover, a variety of cytotoxic mechanism such as apoptotic induction and inhibition of tubulin polymerization have been postulated as a plausible mechanism responsible for anticancer activity of cytotoxic quinolones (11-13).
According to above findings and based on ciprofloxacin and norfloxacin core ( Figure  1), several novel quinolone derivatives were synthesized and displayed significant anticancer activity. Structure-activity relationship study of cytotoxic quinolones demonstrated that substitution of aromatic moieties at C-7 position of quinolone central nucleus mainly affect the anticancer activity of these compounds and enhances selectivity toward type II of human topoisomerase rather than bacterial type (9, 14, 15). Chemical structure of some potent cytotoxic quinolones containing aromatic and heteroaromatic substitutes at C-7 position such as 7-pyridinyl (WIN57294) and 7-hydroxyphenyl 953) are demonstrated in Figure 1

Cytotoxic evaluation
The synthesized compounds 7a-i were tested against different human breast tumor cell lines including MCF-7, MDA-MB-231 and T47D using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) reduction assay. The cell lines were purchased from National Cell Bank of Iran (NCBI). Cells were seeded in 96well plates at the density of 10,000 viable cells per well and incubated at 37º C in a humidified atmosphere with 5% CO 2 for 24 h to allow cell attachment. The cells were then incubated for another 48 h with various concentrations of compounds 7a-i. The synthetic compounds were dissolved in DMSO and the final concentration of DMSO in each well was kept below 1%. etoposide was used as a positive control for each cell line. The medium was replaced with 200 µL RPMI-1640 without phenol red containing 0.5 mg/mL MTT. An additional 4h of incubation at 37 °C were done and then the medium was discarded. Dimethyl sulfoxide (100 μL) was added to each well and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The inhibitory concentration (IC 50 ) was determined and reported in (1) (3) (8) of these series (the corresponding IC 50 values against MCF-7, MDA-MB-231 and T47D cells were 3.03, 11.90 and 2.20 μM, respectively). While its cyclopropyl quinolone counterpart 7h was inactive against MCF-7 and MDA-MB-231 cancer cells (IC 50 >100 μM) and showed moderate cytotoxic potential against T47D cells (IC 50 =12.35μM).

Conclusion
In conclusion, a novel series of 6-fluoro quinolone 3-carboxylic acid and 6-fluroro-1,8-naphthyridone 3-carboxylic acid derivatives containing N-2-(2-furyl)-2-(chlorobenzyloxyimino)ethyl or N-2-(3-furyl)-2-(chlorobenzyloxyimino)ethyl piperazinyl pendant attached to the central core was synthesized and evaluated against three different human cancer cell lines. The most promising compound of ethyl quinolone series, 7 g, demonstrated significant growth inhibitory potential against all evaluated cell lines. The results of structure-activity relationship study demonstrated that quinolone derivatives are superior in cytotoxic potential compared to 1,8-naphthyridone series. Furthermore, ethyl quinolone derivatives were more potent cytotoxic agents than cyclopropyl quinolones.